Progeny from irradiated colorectal cancer cells acquire an EMT-like phenotype and activate Wnt/ß-catenin pathway.

Radiotherapy remains a major approach to adjuvant therapy for patients with advanced colorectal cancer, however, the fractionation schedules frequently allow for the repopulation of surviving tumors cells, neoplastic progression, and subsequent metastasis. The aim of the present study was to analyze the transgenerational effects induced by radiation and evaluate whether it could increase the malignant features on the progeny derived from irradiated parental colorectal cancer cells, Caco-2, HT-29, and HCT-116. The progeny of these cells displayed a differential radioresistance as seen by clonogenic and caspase activation assay and had a direct correlation with survivin expression as observed by immunoblotting. Immunofluorescence showed that the most radioresistant progenies had an aberrant morphology, disturbance of the cell-cell adhesion contacts, disorganization of the actin cytoskeleton, and vimentin filaments. Only the progeny derived from intermediary radioresistant cells, HT-29, reduced the E-cadherin expression and overexpressed ß-catenin and vimentin with increased cell migration, invasion, and metalloprotease activation as seen by immunoblotting, wound healing, invasion, and metalloprotease activity assay. We also observed that this most aggressive progeny increased the Wnt/ß-catenin-dependent TCF/LEF activity and underwent an upregulation of mesenchymal markers and downregulation of E-cadherin, as determined by qRT-PCR. Our results showed that the intermediate radioresistant cells can generate more aggressive cellular progeny with the EMT-like phenotype. The Wnt/ß-catenin pathway may constitute an important target for new adjuvant treatment schedules with radiotherapy, with the goal of reducing the migratory and invasive potential of the remaining cells after treatment.

Blockade of irradiation-induced autophagosome formation impairs proliferation but does not enhance cell death in HCT-116 human colorectal carcinoma cells.

This work was undertaken to gain further information on the molecular mechanisms underlying autophagosome formation and its relation with tumor cell survival in response to radiation in colon cancer. A human colon cancer cell line, HCT-116, was examined with respect to cell survival after blockade of irradiation-induced autophagosome formation by pharmacological interference. Autophagosome formation was confirmed using a kinetic study with incorporated bovine serum albumin gold-conjugate (BSA-Au) analyzed by electron microscopy and an autophagosome-associated LC3B antibody measured by immunofluorescence and Western blotting. Annexin V/PI double staining was used to monitor cell death by apoptosis, and cell cycle profiles by flow cytometry. Ionizing radiation (IR) promoted autophagosome formation in the HCT-116 IR-surviving cells. Pharmacological interference showed that PI3K/Akt and Src were involved in early stages of autophagosome formation. IR alone decreased cell proliferation by arresting cells in the G2/M phase, and pharmacological interference of autophagosome formation decreased proliferation, but did not affect cell survival. Also, our data suggest that decreased proliferation caused by PI3K and Src inhibitors could be through S phase cell cycle delay. Our results clearly indicate that blockade of IR-induced autophagosome formation impairs proliferation but does not enhance cell death in colon cancer cells.

Lysophosphatidic acid induces a migratory phenotype through a crosstalk between RhoA-Rock and Src-FAK signalling in colon cancer cells.

Lysophosphatidic acid (LPA) acts as a potent stimulator of tumorigenesis. Cell-cell adhesion disassembly, actin cytoskeletal alterations, and increased migratory potential are initial steps of colorectal cancer progression. However, the role that LPA plays in these events in this cancer type is still unknown. We explored this question by using Caco-2 cells, as colon cancer model, and treatment with LPA or pretreatment with different cell signalling inhibitors. Changes in the location of adherent junction proteins were examined by immunofluorescence and immunoblotting. The actin cytoskeleton organisation and focal adhesion were analysed by confocal microscopy. Rho-GTPase activation was analysed by the pull-down assay, FAK and Src activation by immunoblotting, and cell migration by the wound healing technique. We show that LPA induced adherent junction disassembly, perijunctional actin cytoskeletal reorganisation, and increased cell migration. These events were dependent on Src, Rho and Rock because their chemical inhibitors PP2, toxin A and Y27632, respectively, abrogated the effects of LPA. Moreover, we showed that Src acts upstream of RhoA in this signalling cascade and that LPA induces focal adhesion formation and FAK redistribution and activation in confluent monolayers. Focal adhesion formation was also observed in the front of migrating cells in response to LPA, and Rock inhibitor abolished this effect. In conclusion, our findings show that LPA modulates adherent junction disassembly, actin cytoskeletal disorganisation, and focal adhesion formation, conferring a migratory phenotype in colon tumour cells. We suggest a functional regulatory cascade that integrates RhoA-Rock and Src-FAK signalling to control these events during colorectal cancer progression.

Lithium reduces tumorigenic potential in response to EGF signaling in human colorectal cancer cells.

Lithium is a specific inhibitor of GSK3-ß, and hence, an activator of the Wnt/ß-catenin pathway, whereas the epidermal growth factor (EGF) has been linked to malignant transformation in epithelial cancer cells. Both pathways are aberrantly activated in most colorectal cancers (CRCs). However, the relationship between them in modulating events related to the progression of this cancer type remains to be defined. In this study, we investigated whether the Wnt/ß-catenin and EGFR pathways converge to modulate the malignant potential of CRC. We used Caco-2 cells, a well-established model used to study CRC, and treatments with lithium chloride, as a modulator of the Wnt/ß-catenin pathway, and with EGF as an inducer of EGFR signaling. We found that both agents altered the subcellular distribution of claudin-1 and ß-catenin, two important proteins of the apical junctional complex, but not their abundance in the cell. Nuclear stabilization of ß-catenin, a marker of Wnt pathway activation, was observed after treatment with both compounds. However, lithium, but not EGF, inhibited GSK3-ß, indicating that these agents modulate this enzyme in a differential fashion. Furthermore, EGF treatment increased the proliferative and migratory capacity but did not alter the colony formation potential of these cells. Surprisingly, lithium, known to activate the Wnt/ß-catenin pathway, inhibited the increased proliferation by arresting cells in the G2/M phase as well as the cell migration promoted by EGF, as demonstrated by the combined treatment with these agents. Lithium treatment alone reduced the cell colony formation. Thus, our findings suggest that lithium plays an important role in regulating cellular events related to tumor progression in CRC.

Serine protease activities in Leishmania (Leishmania) chagasi promastigotes.

The present work reports the isolation, biochemical characterization, and subcellular location of serine proteases from aqueous, detergent soluble, and culture supernatant of Leishmania chagasi promastigote extracts, respectively, LCSII, LCSI, and LCSIII. The active enzyme molecular masses of LCSII were about 105, 66, and 60 kDa; of LCSI, 60 and 58 kDa; and of LCSIII, approximately 76 and 68 kDa. Optimal pH for the enzymes was 7.0 for LCSI and LCSIII and 8.5 for LCSII, and the optimal temperature for all enzymes was 37°C, using α-N-ρ-tosyl-L: -arginine methyl ester as substrate. Assay of thermal stability indicated that LCSIII is the more stable enzyme. Hemoglobin, bovine serum albumin, and ovalbumin were hydrolyzed by LCSII and LCSI but not by LCSIII. Inhibition studies suggested that enzymes belong to the serine protease class modulated by divalent cations. Rabbit antiserum against 56-kDa serine protease of Leishmania amazonensis identified proteins in all extracts of L. chagasi. Furthermore, immunocytochemistry demonstrated that serine proteases are located in flagellar pocket region and cytoplasmic vesicles of L. chagasi promastigotes. These findings indicate that L. chagasi serine proteases differ from L. amazonensis proteases and all known flagellate proteases, but display some similarities with serine proteases from other Leishmania species, suggesting a conservation of this enzymatic activity in the genus.

Mecanismos celulares e moleculares modulados por PGE2 e TGFβ durante a progressão do câncer colorretal

Um dos primeiros eventos associados à progressão do câncer colorretal (CCR) é a perda dos contatos célula-célula, seguido da aquisição de um fenótipo indiferenciado e aumento na motilidade celular. Estes eventos estão associados com um processo conhecido como transição epitélio-mesenquimal (TEM), na qual as células adquirem capacidade para invadir tecidos vizinhos. A biogênese deste tipo de câncer envolve uma série de fatores predisponentes dentre os quais está o processo infamatório, onde a prostaglandina E2 (PGE2) e o fator transformador β de crescimento (TGFβ) cumprem importante função. Vários estudos têm mostrado que a PGE2 induz proliferação, migração e invasão celular. Já o TGFβ, apesar de ter uma função dual como promotor ou supressor da tumorigênese, é conhecido também por promover TEM em diferentes tipos de câncer. No entanto, a associação entre as vias de sinalização celular ativada por estes agentes na indução da perda da adesão célula-célula e da TEM em CCR não é conhecida. No presente estudo, usando linhagens celulares de CCR, analisamos a associação entre a PGE2 e o TGFβ sobre as alterações no fenótipo celular e eventos relacionados com a TEM. Na primeira etapa do estudo, usando células Caco-2, mostramos que o tratamento com PGE2 promoveu alterações na localização e função de proteínas do complexo juncional apical (CJA) concomitantemente com a perda da funcionalidade da barreira paracelular. Mostramos também que a via de sinalização responsável nesse evento foi a PKC através de um mecanismo envolvendo os receptores prostanóides EP1 e EP2 e a proteína claudina-1 na modulação do CJA. Na segunda etapa do estudo, analisamos eventos relacionados com a TEM usando linhagens celulares de CCR que diferem no fenótipo e no grau de invasão mediante tratamentos com PGE2 e TGFβ...

The lost of cell-cell adhesion followed by increased motility and acquisition of an undifferentiated phenotype are initial events associated with colorectal cancer (CCR) progression. These events are associated with a process known as epithelial-mesenchymal transition (TEM) in which cells acquire the ability to invade neighboring tissues. The biogenesis of this type of cancer predisposes a series of conditions for which the inflammatory process, known as an important inductor of prostaglandin E2 (PGE2) and tumor growth factor β (TGFβ), plays an important function. Various studies have shown that PGE2 is able to induce proliferation, migration and invasion. TGFβ plays a dual function either as promoter or tumor suppressor and is also a TEM inductor in different types of cancer. However, a crosslink between the cell signaling pathways activated by these agents in relation to lost of cell-cell adhesion and TEM development in CCR is unknown. In the present study, using CCR cell lines, we analyze the association between PGE2 and TGFβ...

Modulação do processo de adesão célula-célula por prostaglandina E2 em células de adenocarcinoma de cólon humano / Modulation of the adhesion process cell-cell for prostaglandim E2 in cells of human colon adenocarcinoma

A enzima ciclooxigenase-2 juntamente com a prostaglandina E2 (PGE2), tem sido descrito como potentes indutores da proliferação, migração e invasividade celular em câncer colo-retal. Neste trabalho nós investigamos os efeitos do tratamento da PGE2 no complexo juncional (CJ) de células epiteliais derivadas de adenocarcinoma de cólon humano, Caco-2. As células foram tratadas com PGE2 (1 μM) e os efeitos no complexo juncional foram monitorados em diferentes tempos de tratamento. Alterações nas proteínas de junção tight e aderente foram observadas pela medida da resistência elétrica transepitelial (TER), por imunofluorescência e immunoblotting e microscopia eletrônica de transmissão (TEM). Nossos resultados mostraram que células tratadas por 15 minutos com PGE2 (1 μM) apresentaram uma diminuição de aproximadamente 40por cento na TER. Quando a monocamada de células foi analisada por MET, foi possível observar grandes espaços na região das junções aderentes nas células tratadas durante 15, 30 e 60 minutos quando comparada a células não tratadas, que apresentaram um CJ bem definido. As juncões tight apareceram aparentemente preservadas durante os tempos de tratamento utilizados com PGE2. No entanto, usando a técnica do vermelho de rutênio, mostramos que as junções tight não estavam funcionais como verificado pela passagem dessas partículas através do espaço intercelular. Alterações no perfil de distribuição das proteínas do complexo juncional foram observadas para E-caderina, β-catenina, ocludina, ZO-1 e claudinas –1 e –4, principalmente nos primeiros minutos de tratamento. O presente estudo mostra que a PGE2 causa alterações no complexo juncional, provocando alterações da permeabilidade paracelular a íons e moléculas, rompimento do contato célula-célula e redistribuição das principais proteínas das junções tight e aderentes em células Caco-2.

Modulação dp processo de adesão célula-célula por prostaglandina E2 em células de adenocarcinoma de cólon humano

A enzima ciclooxigenase-2 juntamente com a prostaglandina E2 (PGE2), tem sido descrito como potentes indutores da proliferação, migração e invasividade celular em câncer colo-retal. Neste trabalho nós investigamos os efeitos do tratamento da PGE2 no complexo juncional (CJ) de células epiteliais derivadas de adenocarcinoma de cólon humano, Caco-2. As células foram tratadas com PGE2 (1 μM) e os efeitos no complexo juncional foram monitorados em diferentes tempos de tratamento. Alterações nas proteínas de junção tight e aderente foram observadas pela medida da resistência elétrica transepitelial (TER), por imunofluorescência e immunoblotting e microscopia eletrônica de transmissão (TEM). Nossos resultados mostraram que células tratadas por 15 minutos com PGE2 (1 μM) apresentaram uma diminuição de aproximadamente 40por cento na TER. Quando a monocamada de células foi analisada por MET, foi possível observar grandes espaços na região das junções aderentes nas células tratadas durante 15, 30 e 60 minutos quando comparada a células não tratadas, que apresentaram um CJ bem definido. As juncões tight apareceram aparentemente preservadas durante os tempos de tratamento utilizados com PGE2. No entanto, usando a técnica do vermelho de rutênio, mostramos que as junções tight não estavam funcionais como verificado pela passagem dessas partículas através do espaço intercelular. Alterações no perfil de distribuição das proteínas do complexo juncional foram observadas para E-caderina, β-catenina, ocludina, ZO-1 e claudinas û1 e û4, principalmente nos primeiros minutos de tratamento. O presente estudo mostra que a PGE2 causa alterações no complexo juncional, provocando alterações da permeabilidade paracelular a íons e moléculas, rompimento do contato célula-célula e redistribuição das principais proteínas das junções tight e aderentes em células Caco-2.